Fig 1: Cells carrying the uL6 p.Leu20Pro variant reveal translational fidelity defects. (A) Cell-based assay to measure UAG and UGA stop codon readthough using bicistronic luciferase reporters transfected in LCLs derived from individuals carrying the RPL9 c.-2+1G>C variant (P2), the uL6 p.Leu20Pro variant (P3), or unrelated healthy controls. *P < 0.05, **P < 0.01. (B) Cell-free assay measuring the level of UAG stop codon readthrough in ribosomes purified from LCLs carrying the RPL9 c.-2+1G>C (P2) or uL6 p.Leu20Pro (P3) variant compared to LCLs from three unrelated healthy controls. Experiments were performed three times in duplicate with two different preparations of ribosomes per cell type; error bars represent standard error. To determine statistical significance, a paired Student's t-test was applied. **P < 0.01. (C) MS-based proteomic characterization of ribosomes purified from LCLs of P2 carrying the uL6 p.Leu20Pro variant (N = 3). Sequences and scores of peptides identified by Mascot search engine and covering the position 20 of uL6 are presented. On the y-axis the amino acid in position 20 of uL6 is in bold font and underlined. (D) Quantification of the abundance of uL6 peptides carrying Pro20 or Leu20 measured in purified ribosomes from LCLs generated from a healthy control or P2. (E) Predicted structure of uL6 p.Leu20Pro variant protein (in pink) superimposed on the known structure of wild type uL6 (in lime green). Helix 95 of the SRL is shown in gold, amino acid 20 (aa20) is shown in red, rRNA helix 97 is shown in cyan, rRNA extension segment 39 (ES39) is shown in light purple, eEF2 is shown in dark purple, and the positions of the Lys21 (wild type in lime green, variant in pink) are indicated with arrows.
Fig 2: Metabolic profiles and enrichment analysis of LCLs carrying RPL9 variants. (A) Metabolic profile heat map showing Z-scores of LCLs derived from three unrelated healthy controls compared to cells from DBA-affected individual (P1) carrying variants in the 5′UTR of RPL9. (B) Enrichment analysis (using Metabolanalyst 3.0 online software) of significantly changed metabolites from (A). (C) Metabolic profile heat map showing Z-scores of LCLs derived from three unrelated healthy controls compared to cells from an individual carrying the uL6 (RPL9) p.Leu20Pro variant (P2). (D) Enrichment analysis of significantly changed metabolites from (B). For (A) and (C) only metabolites with comparative VIP-Scores >1 (out of 63 metabolites measured) are listed.
Fig 3: Variants in RPL9 confer different polysome profile peak ratios. (A–C) Representative polysome profiles of LCLs derived from a healthy control (A), a DBA-affected individual (P1) carrying the RPL9 c.-2+1 variant (B), and an individual (P2) carrying the uL6 p.Leu20Pro variant (C). or healthy controls. The free 40S and 60S subunits, 80S monosomes, and polysomes are labeled. The reduced 60S peaks in the profiles are indicated with open arrows, the reduced 80S monosomes indicated by filled arrows.
Fig 4: Erythroid cell culture assays of primary CD34+ cells reveal proliferation defects only in cells with 5′UTR variants. (A) Growth curves of CD34+ cells isolated from peripheral blood of the DBA-affected individual carrying the RPL9 c.-2+1 variant (P1, red) compared to erythroid cells from a healthy control (blue). (B) Western blotting of lysates from cells in (A) collected at Day 7 and probed with antibodies against uL6 protein. (C) Growth curves of CD34+ cells isolated from the individual carrying the uL6 (RPL9) p.Leu20Pro variant (P2, red) compared to cells from a healthy control (blue). (D) Western blotting of lysates from cells in (C) collected at Day 7 and probed with antibodies against uL6 protein.
Fig 5: Variants in RPL9 recapitulate specific pre-rRNA processing defects found in RP depleted cells and show key differences. (A) Northern blot analysis of LCLs derived from individuals carrying RPL9 variants. Radio-labeled probes against ITS2 (left panels), ITS1–5.8S (upper right panels), 5′ITS1 (middle panels), or 18S and 28S (lower right panels) rRNA sequences were used to blot 3 μg total RNA isolated from cells. (B) Quantification of three independent experiments analyzing rRNA precursors in LCLs derived from individuals carrying RPL9 variants using RAMP.
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